Diverse effects of monensin on capacitative Ca2+ entry and release of

stored Ca2+ in vascular smooth muscle cells

Ichiro Wakabayashi*, Mikio Marumo, Yoko Sotoda

Department of Hygiene and Preventive Medicine, School of Medicine, Yamagata University, Iida-Nishi 2-2-2, Yamagata 990-9585, Japan

Received 2 December 2002; received in revised form 15 January 2003; accepted 24 January 2003

Abstract

The effects of monensin, an activator of Na+/H+ exchanger (NHE), on capacitative Ca2 + entry (CCE) were investigated using A7r5 cells.

Capacitative Ca2 + entry was induced by elevation of extracellular Ca2 + concentrations of A7r5 cells in which stored Ca2 + had been depleted

by previous administration of thapsigargin. Capacitative Ca2 + entry was abolished by pretreatment of the cells with SKF-96365 (1-[h-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) but was not affected by pretreatment with verapamil.

Monensin significantly increased capacitative Ca2 + entry. On the other hand, 5-hydroxytryptamine-induced inositol monophosphate

accumulation and subsequent intracellular Ca2 + release from its stores were significantly inhibited by monensin, while thapsigargin-induced

Ca2 + release was not affected by monensin. These results suggest that monensin has diverse actions on capacitative Ca2 + entry and agonist-

induced release of stored Ca2 + in vascular smooth muscle cells.

D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Ca2+ channel; Ca2+ entry, capacitative; Inositol 1,4,5-trisphosphate; Monensin; Smooth muscle, vascular; Vasoconstriction

1. Introduction

Phosphoinositide hydrolysis is an initial response follow-

ing stimulation of plasmalemmal receptors by G protein-

coupled receptor agonists and triggers cellular activation in a

variety of cells, including vascular smooth muscle cells

(Abdel-Latif, 1986). Inositol trisphosphate (IP3), a metabo-

lite of phosphoinositide hydrolysis, induces release of Ca2 +

from its intracellular stores. The depletion of Ca2 + stores

then induces voltage-independent plasmalemmal Ca2 + entry,

known as capacitative Ca2 + entry (CCE) (Putney, 1986).

Thus, CCE is thought to be involved in a mechanism of

receptor-stimulated Ca2 + entry into vascular smooth muscle

cells (Putney, 1997; McFadzean and Gibson, 2002). Diac-

ylglycerol, another metabolite of phosphoinositide hydroly-

sis, activates protein kinase C (PKC), which plays crucial

roles in cellular functions. In vascular smooth muscle cells,

PKC activation results in activation of plasmalemmal volt-

age-dependent Ca2 + channels (Gollasch and Nelson, 1997)

as well as increase in Ca2 + sensitivity of the contractile

apparatus (Rasmussen et al., 1987). However, the mecha-

nism of Ca2 + channel opening following PKC activation is

still not clear. On the other hand, PKC is known to activate

the Na+/H+ exchanger (NHE), which is a ubiquitously

expressed membrane transport protein, through phosphory-

lation of its serine residues (Sardet et al., 1990). In vascular

smooth muscle cells, receptor stimulation by agonists such as

angiotensin II results in activation of NHE, which is involved

in a mechanism of proliferative action of the agonists (Berk

et al., 1987; LaPointe and Batlle, 1994). However, it is not

known whether modulation of NHE affects the mechanism

of Ca2 + entry, including CCE, into vascular smooth muscle

cells. In the present study, we therefore investigated the

effects of monensin, an activator of NHE, on CCE and its

related mechanisms such as phosphoinositide hydrolysis and

Ca2 + release from its stores. CCE was induced by elevation

of extracellular Ca2 + concentrations in the presence of

thapsigargin, a Ca2 +-ATPase inhibitor (Treiman et al.,

1998), and phosphoinositide hydrolysis was evaluated by

accumulation of inositol monophosphate in the presence of

LiCl (Berridge et al., 1982).

0014-2999/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0014-2999(03)01372-4

* Corresponding author. Tel.: +81-23-628-5252; fax: +81-23-628-

5255.

E-mail address: wakabaya@med.id.yamagata-u.ac.jp (I. Wakabayashi).

www.elsevier.com/locate/ejphar

European Journal of Pharmacology 464 (2003) 27–31

2. Materials and methods

2.1. Cell culture

A7r5 rat aortic smooth muscle cells were obtained from

Dainippon Pharmaceutical (Osaka, Japan) and were cultured

in Dulbecco’s Modified Eagle’s Medium (DMEM) contain-

ing 5% fetal calf serum, 4 mM glutamate, 100 U/ml

penicillin, 100 Ag/ml streptomycin and 0.25 Ag/ml ampho-

tericin B in a humidified atmosphere at 37 jC under 5%

CO2–95% air. The cells were spread in 12-well or 100-mm

dish and cultured until reaching a confluent condition. Then,

confluent cells were used for the assays.

2.2. Measurement of intracellular free Ca2+ concentration

[Ca2 +]i (intracellular free Ca2 + concentration) was meas-

ured using a fluorescent Ca2 + indicator, fura-2. A7r5 cells

were loaded with fura-2/acetoxymethyl ester (AM) (final

concentration, 5 AM) at 37 jC for 30 min. After loading, the

cells were washed once with physiological salt solution

(PSS) buffered byHEPES (mM:NaCl 135,KCl 5,KH2PO41,

CaCl2 2.5,MgCl2 1, HEPES 10, glucose 10, pH 7.4), and they

were resuspended in Ca2 +-free PSS buffered by HEPES and

containing 0.01 mM EGTA (nominally Ca2 +-free solution).

Fluorescence measurements were carried out with a dual-

wavelength spectrofluorometer (F2500 Fluorescence Spec-

trophotometer, Hitachi, Tokyo, Japan) using a 0.4-ml cuvette

maintained at 37 jC. The wavelengths used for excitation

were 340 and 380 nm, and the wavelength used for emission

was 510 nm. Using a ratio (R) of fluorescence intensity (F)

of F340/F380, the fractional changes in [Ca2 +]i were deter-

mined. The fluorescence after sequential addition of 0.2%

Triton X-100 and EGTA (5 mM) to the cuvette provided the

maximum fluorescence ratio (Rmax) and minimum fluores-

cence ratio (Rmin), respectively. [Ca2 +]i was calculated using

the formula described by Grynkiewicz et al. (1985):

½Ca2þ�i ¼ ðR� RminÞ=ðRmax � RÞ � b � Kd;

where b is the ratio of the emission fluorescence values at

380 nm excitation in the presence of Triton X-100 and

EGTA, and Kd, the dissociation constant for Ca2 +, is 224.

CCE was expressed as a net increase in [Ca2 +]i evoked by

extracellular addition of CaCl2 following thapsigargin stim-

ulation in nominally Ca2 +-free medium.

2.3. Measurement of inositol monophosphate accumulation

The A7r5 cells were stabilized in each well containing

0.5 ml of DMEM, and then myo-[2-3H]inositol (0.05 AM)

was added to the medium. After 24 h of incubation, the cells

were used for measurement of inositol monophosphate

accumulation. Inositol monophosphate accumulation was

measured according to the method described previously

(Berridge et al., 1982). The cells were rinsed three times

with fresh warm (37 jC) phosphate-buffered saline. Then,

LiCl at a final concentration of 10 mM was added to each

well containing 0.5 ml of DMEM, and 5-hydroxytryptamine

(5-HT, 100 AM) or the vehicle was added to the well 30 min

later and further incubated at 37 jC for 20 min. The reaction

was then terminated by addition of 0.9 ml of chloroform–

methanol solution (1:2, v/v), followed by addition of 0.3 ml

of chloroform and vortexing. Water (0.3 ml) was then

added, followed by vigorous vortexing. The medium in

each well was collected in a glass tube and then centrifuged

at 1000� g for 5 min, allowing the aqueous and chloroform

phases to separate. An aliquot of 0.9 ml of the upper phase

was then loaded onto AG 1X8 (formate form) resin packed

in a disposable column. The columns were then sequentially

washed with 9 ml of water, 9 ml of 60 mM sodium formate/

5 mM sodium borate, and 9 ml of 1 M ammonium formate/

0.1 M formic acid to selectively elute [3H]inositol mono-

phosphate. Aliquots (3 ml) of the eluant were mixed with

scintillant, and its radioactivity was counted in a liquid

scintillation spectrophotometer. The level of inositol mono-

phosphate accumulation was expressed as [3H]inositol

monophosphate level (dpm) of each aliquot.

2.4. Drugs

Fura-2/AM (Dojin, Kumamoto, Japan) and thapsigargin

(Sigma, St. Louis, MO, USA) were dissolved in dimethyl-

sulfoxide to make a stock solutions of 5 mM and 1 mM,

respectively, and stored at � 30 jC. 1-[h-(3-[4-Methoxy-

phenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole

hydrochloride (SKF-96365, Calbiochem, La Jolla, CA,

USA) was dissolved in distilled water to make a stock

solution of 10 mM and stored at � 30 jC. Verapamil

(Wako, Osaka, Japan) and 5-hydroxytryptamine hydrochlor-

ide (Sigma) were dissolved in distilled water to make stock

solutions of 10 mM, and stored at 4 jC. Monensin (Sigma)

was dissolved in ethanol to make a stock solution of 50 mM,

which was stored at � 30 jC and diluted with distilled

water just before use. (1-[6-([(17h)-3-Methoxyestra-

1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione)

(U73122, Sigma) was dissolved in dimethylsulfoxide to

make a stock solution of 1 mM, which was stored at

� 30 jC and diluted with distilled water just before use.

2.5. Statistical analysis

Statistical analysis was done using Student’s t test or

Mann–Whitney U test. P values less than 0.05 were

regarded as significant.

3. Results

3.1. Effects of monensin on thapsigargin-induced CCE

Fig. 1A shows a representative recording of CCE induced

in the presence of thapsigargin in A7r5 cells. In nominally

I. Wakabayashi et al. / European Journal of Pharmacology 464 (2003) 27–3128

Ca2 +-free solution, the addition of thapsigargin (0.1 AM)

resulted in an increase in [Ca2 +]i. Then elevation of the Ca2 +

concentration of the medium from nominal zero to 1.5 mM

resulted in a further increase in [Ca2 +]i (CCE). The CCE was

strongly inhibited by pretreatment with SKF-96365 but was

not affected by pretreatment with verapamil (Fig. 1B).

Pretreatment of the cells with monensin (1 and 10 AM)

augmented significantly the CCE (Fig. 1) in a concentration-

dependent manner (Fig. 2). SKF-96365 abolished the CCE

and the enhancing effect of monensin, but verapamil did not

affect them (Fig. 1B).

3.2. Effects of monensin on thapsigargin- and 5-HT-induced

Ca2+ release from intracellular Ca2+ stores

In nominally Ca2 +-free solution, stimulation of A7r5

cells with thapsigargin (0.1 AM) and 5-HT (100 AM) caused

Fig. 1. (A) Representative recording of induction of capacitative Ca2 + entry

inA7r5 cells. The cells were incubated in nominally Ca2 +-free solution. After

stabilization, the cells were stimulated with thapsigargin (0.1 AM). At 3 min

after thapsigargin stimulation, CaCl2 (1.5 mM) was added to the cuvette

containing the cells. Monensin (10 AM) or a vehicle was added to the cuvette

at 2 min before addition of CaCl2. (B) Effects of monensin on capacitative

Ca2 + entry in A7r5 cells. In nominally Ca2 +-free solution, A7r5 cells were

incubated with thapsigargin (0.1 AM) for 3 min; then CaCl2 (1.5 mM) was

added to the solution. Monensin (10 AM) or a vehicle was added to the

solution at 2 min before addition of Ca2 +. In some experiments, SKF-96365

(100 AM) or verapamil (1 AM) was added to the solution at 1 min before

addition of Ca2 +. [Ca2 +]i at the peak after addition of Ca2 + was used for the

analysis. Asterisks denote significant differences from the condition without

pretreatment with monensin (*) and from the condition without pretreatment

with SKF-96365 (**). NS, not significant difference. n= 4–5.

Fig. 2. Effects of monensin on thapsigargin-induced capacitative Ca2 + entry

(CCE) and 5-hydroxytryptamine (5-HT)-induced increase in [Ca2 +]i (Ca2 +

release) in A7r5 cells. In the experiments for thapsigargin-induced CCE,

A7r5 cells were incubated in nominally Ca2 +-free solution with thap-

sigargin (0.1 AM) for 3 min; then CaCl2 (1.5 mM) was added to the

solution. Monensin (0.1, 1, 10 AM) or a vehicle was added to the solution at

2 min before addition of Ca2 +. [Ca2 +]i at the peak after addition of Ca2 +

was used for the analysis. In the experiments for 5-HT-induced Ca2 +

release, A7r5 cells were incubated with monensin (0.1, 1, 10 AM) for 4 min.

5-HT (100 AM) was then added to the solution. [Ca2 +]i at the peak after

addition of 5-HT was used for the analysis. The data are expressed as the

percentage of CCE or 5-HT-induced Ca2 + release in the cells treated with a

vehicle instead of monensin. Asterisks denote significant differences from

the condition without pretreatment with monensin. n= 5.

Fig. 3. Effects of monensin (A) and SKF-96365 (B) on thapsigargin-or 5-

hydroxytryptamine (5-HT)-induced increases in [Ca2 +]i of A7r5 cells in

Ca2 +-free solution. In nominally Ca2 +-free solution, A7r5 cells were

incubated with monensin (10 AM) or SKF-96365 (100 AM) for 4 min.

Thapsigargin (0.1 AM) or 5-HT (100 AM) was then added to the solution.

[Ca2 +]i at the peak after addition of thapsigargin or 5-HT was used for the

analysis. An asterisk denotes a significant difference compared with the

control pretreated with a vehicle. n= 3–8.

I. Wakabayashi et al. / European Journal of Pharmacology 464 (2003) 27–31 29

a transient increase in [Ca2 +]i. Monensin (1, 10 AM) in-

hibited the 5-HT-induced increase in [Ca2 +]i in a concen-

tration-dependent manner but did not affect the thapsigar-

gin-induced increase in [Ca2 +]i (Figs. 2 and 3A). U73122

(4 AM) abolished 5-HT-induced increase in [Ca2 +]i[82.0F 7.6 nM (control) vs. 7.4F 2.0 nM (U73122-treated)

(P < 0.01)]. Neither thapsigargin-induced nor 5-HT-induced

Ca2 + release was affected by SKF-96365 (Fig. 3B).

3.3. Effects of monensin on 5-HT-induced phosphoinositide

hydrolysis

Inositol monophosphate accumulation was increased by

5-HT (100 AM) stimulation to a level about 2.5 times higher

than the basal level. 5-HT-induced inositol monophosphate

accumulation was significantly inhibited by monensin

(10 AM), while monensin did not affect the basal level of

inositol monophosphate accumulation (Fig. 4).

4. Discussion

The relationship between NHE activation and CCE, both

of which are triggered by accelerated phosphoinositide

turnover following agonist stimulation, has not been clari-

fied. The present study has shown that activation of NHE by

monensin increases thapsigargin-induced voltage-independ-

ent Ca2 + entry. Since CCE and NHE are activated by IP3(through Ca2 + store depletion) and diacylglycerol (through

PKC activation), respectively, following agonist-stimulated

phosphoinositide turnover, it is possible that these two

signal pathways work in harmony to increase Ca2 + entry

due to Ca2 + store depletion. In the present study, monensin

was added to the medium after Ca2 + stores had been

depleted by thapsigargin. Thus, the pathway of CCE after

Ca2 + store depletion may be facilitated by activation of

NHE.

Monensin, which activates NHE, enhanced CCE in A7r5

cells, implying that activation of NHE by monensin leads to

intracellular alkalinization by extrusion of intracellular pro-

tons via NHE. In support of this postulate, we recently

found that monensin induced a gradual increase in intra-

cellular pH by about 0.1 pH when elevation of [Ca2 +]i due

to CCE reached a peak level in the same condition (Waka-

bayashi et al., 2002, unpublished observation). SKF-96365,

an inhibitor of receptor-operated Ca2 + channels (Merritt et

al., 1990), abolished the CCE and the enhancing effect of

monensin, but verapamil, an inhibitor of voltage-dependent

Ca2 + channels, did not affect them. We also found that

phenylephrine-induced verapamil-insensitive Ca2 + entry

and contraction of vascular smooth muscle were enhanced

by NH4Cl-induced increase in intracellular pH (Wakabaya-

shi et al., 2001). Further study is required to elucidate the

causal relationship between intracellular pH and CCE in

vascular smooth muscle. Alternatively, it is possible that the

enhancing effect of monensin on CCE is due to an increase

in Na+ influx by NHE activation and a subsequent increase

in Ca2 + influx through the Na+/Ca2 + exchanger (Valant et

al., 1992). This hypothesis should also be examined in a

future study.

Increased NHE activity has been reported in platelets,

leukocytes and erythrocytes from primary hypertensive

patients (Ng et al., 1989; Semplicini et al., 1989; Rosskopf

et al., 1992). The phosphorylation level of NHE protein in

cells from Wistar–Kyoto (WHY) rats was found to be only

half of that in cells from spontaneously hypertensive (SHR)

rats (Siczkowski and Ng, 1996). Moreover, transgenic mice

in which NHE was constitutively overexpressed developed

hypertension, which was not essential but salt-sensitive

(Kuro-o et al., 1995). These findings suggest that NHE

plays a significant role, especially in the pathogenesis of

salt-sensitive hypertension. Therefore, facilitation of CCE

due to increased NHE activity may be, in part, involved in

increased contractility of VSMC in primary hypertension.

On the other hand, monensin attenuated 5-HT-stimulated

inositol monophosphate accumulation and subsequent

release of Ca2 + from the stores, while thapsigargin-induced

Ca2 + release was not affected by monensin. 5-HT-induced

Ca2 + release was dependent on phosphoinositide hydroly-

sis, since U73122, a phospholipase C inhibitor, abolished 5-

HT-induced increase in [Ca2 +]i of A7r5 cells. These find-

ings suggest that activation of NHE inhibits agonist-induced

phosphoinositide hydrolysis, resulting in a decrease in IP3-

induced Ca2 + release from the stores. The concentration

dependence of both of the diverse effects of monensin on

CCE and 5-HT-induced Ca2 + release supports the specula-

tion that the mechanisms underlying these effects of mon-

ensin are the same. In the present study, monensin was used

as a stimulant of Na+/H+ exchanger, which is activated by

PKC following diacylglycerol formation. Monensin

inhibited 5-HT-induced acceleration of phosphoinositide

hydrolysis and thereby the 5-HT-induced Ca2 + release.

Thus, activation of the Na+/H+ exchanger following agonist

stimulation may play a negative feedback role in agonist-

induced phosphoinositide hydrolysis. There have been sev-

Fig. 4. Effects of monensin on 5-hydroxytryptamine (5-HT)-induced

inositol monophosphate accumulation in A7r5 cells. A7r5 cells were

pretreated with monensin (10 AM) for 1 min and then stimulated with 5-HT

(100 AM) for 20 min. An asterisk denotes a significant difference compared

with the control pretreated with a vehicle. n= 5.

I. Wakabayashi et al. / European Journal of Pharmacology 464 (2003) 27–3130

eral studies on the effects of monensin on phosphoinositide

hydrolysis, and the results of the previous studies, in

contrast to the results of the present study, have shown that

monensin stimulates phosphoinositide hydrolysis by

increasing intracellular Na+ in brain synaptoneurosomes

(Gusovsky et al., 1987) and microvessels (Catalan et al.,

1996). Thus, the effects of monensin on phosphoinositide

hydrolysis differ depending on the cell type. A7r5 cell is a

cell line originating from fetal rat aortic smooth muscle and

is often used for studies on function of vascular smooth

muscle cells. Capacitative Ca2 + channels have been dem-

onstrated in A7r5 cells that are activated by agonists such as

endothelin-1 (Miwa et al., 1999). However, further studies

using native vascular smooth muscle cells are needed to

clarify the mechanism of the inhibitory action of monensin

on phosphoinositide hydrolysis.

In conclusion, monensin, an activator of NHE, facilitates

CCE and inhibits agonist-induced phosphoinositide hydrol-

ysis and subsequent Ca2 + release from the stores, suggest-

ing that NHE plays crucial roles in agonist-stimulated Ca2 +

entry and its related regulation of signal transduction.

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